Malaria is a severely debilitating, widespread disease caused by infection by a parasite of the genus Plasmodium. Despite years of research and major expenditures for eradication or control, the disease remains a major world-wide public health problem, both for those living in areas where the disease is endemic and for travelers and visitors to such areas. Although once commonly encountered in the continental United States, the disease is now principally found in tropical and semitropical areas of the world including many developing nations.
The major species infective to humans are Plasmodium falciparum and Plasmodium vivax. Two other plasmodial species, Plasmodium ovale and Plasmodium malarie, occur with much less frequency, but the latter one may induce considerable immunopathology, particularly in children. Other species of the parasite are infective in other mammalian species.
The complex life cycle of the parasite is well understood in general outline, although much remains to be understood at the biochemical and genetic levels. In infected humans, the organism exists primarily as an intracellular parasite of red blood cells in which it undergoes vegetative reproduction. There is also a transient extracellular phase during which organisms migrate through the bloodstream to infect new host cells. (The synchronous rupture of large numbers of infected red blood cells, releasing free parasites, is frequently associated with fever). The blood forms of the parasite, both intra- and extra-cellular, are well adapted to the host, and elicit only an inadequate protective antibody response.
Malaria is transmitted by the bite of a mosquito. The mosquito functions as more than a passive vector for the parasite. The Plasmodium undergoes several stages of differentiation in the mosquito host, and also undergoes sexual mating. In the mosquito, the organism is first found in the gut, later in the hemocele of the thoracic region, and finally in the salivary glands from which they are injected into the human or other mammalian host when the mosquito next feeds. The differentiated form found in the salivary gland is referred to as a sporozoite. As the mosquito feeds, sporozoites enter the bloodstream where they exist transiently before entering primary target cells, presumably cells of the liver.
Attempts to control malaria have been partly but not wholly successful. The disease has been largely eliminated from areas where mosquito abatement and eradication programs have succeeded in removing or controlling the population of mosquito vectors. Nevertheless, the disease is still endemic in large portions of the world. No effective means of prevention for individuals who risk exposure to the disease has been found to date except by drug administration.
The major prior art strategy for the control or prevention of malarial infections in individuals has been based upon attempts to provide immunity against the blood forms of the parasite. The approach was considered advantageous because substantial amounts of experimental material could be obtained from in vitro cultures. Furthermore, if antibodies against the blood forms of Plasmodium could be obtained, they could be administered to patently infected individuals to suppress an ongoing infection. However, attempts to isolate and characterize a unique protective antigen from these forms have not been successful. Furthermore, clinical and experimental observations suggest that the blood forms may not be highly immunogenic.
A second prior art strategy, upon which the present invention is based, has been to attempt to develop antibodies capable of inactivating sporozoites, the primary infective agent. The chief disadvantage of this approach is that sporozoites may be obtained only from infected mosquitos, hence are available in extremely limited quantities. Moreover, it was originally assumed that antibodies to sporozoites would not be effective in preventing the establishment of infection. However, continued experimentation has been encouraged by the discovery that sporozoites, in contrast to the blood forms of Plasmodium, are highly antigenic.
The first demonstration that sporozoites were immunogenic and could be used to protect against infection was provided by Mulligan et al, J.Malar.Inst.India, 4, 25 (1941). Injections of killed P. gallinaceum sporozoites were shown to produce active immunization of fowls against avian malaria caused by the same organism. The results of this experiment were generally assumed to apply only in the case of avian malaria. No further studies on sporozoite immunogenicity were done until the pioneering work of co-inventor R. Nussenzweig was begun. In mice it was shown that sporozoites of P. berghei, inactivated by exposure to X-rays or gamma irradiation, could immunize these animals against the disease when challenged by inoculation of active sporozoites, Nussenzweig, R. S., et al., Nature, 216, 160 (1967); Vanderberg, J., et al., J.Parasitol., 54, 1175 (1968); Nussenzweig, R., et al., Mil.Med., 134, 1176 (1979). Immunity was found to be directed solely against sporozoites and the protected animals remained fully susceptible to infection when challenged with blood stages of the same parasite strain. It was also observed that the route of immunization was of primary importance, and that in the absence of adjuvants, a reproducibly high degree of protection could only be obtained upon repeated intravenous immunization, Spitalny, G., et al., Proc.Helminthol.Soc.Wash., 39, 506 (1972), or through the repeated bite of infected irradiated mosquitoes, Vanderberg, J., et al., J.Parasitol., 56, 350 (1970). It is important to note that only the later maturation stage in the mosquito vector (salivary gland sporozoites) was able to produce protective immunity. Antisera against earlier developmental stages, e.g., oocyst sporozoites, did not protect against mature sporozoites found in the mosquito's salivary glands, Vanderberg, J., et al. (1972), supra. The immunity provided by injection with irradiated sporozoites appears to be induced by both humoral and cell-mediated mechanisms. Circulating antibodies against irradiated sporozoites can inactivate non-irradiated sporozoites in vitro, such that they are unable to produce infection when subsequently injected into non-immune mice. Rats and mice readily produce antibodies to sporozoites of Plasmodial species infective to other animals, including P. falciparum and P. vivax, infective to man. However, these antisera do not cross-react with sporozoites of other malarial species and do not protect these animals against rodent infective strains. It has been noted, however, that different geographic isolates of the same species do cross-react extensively.
Sporozoite injection has been demonstrated to provide immunity in primates, i.e., rhesus monkeys inoculated with P. cynomolgi and P.knowlesi. Owing to the greater difficulty and expense of working with such experimental animals, the data relating to immunization of monkeys is necessarily less complete than for rodents. Early attempts to immunize rhesus monkeys against P. cynomolgi failed to demonstrate total protection against sporozoite challenge, Collins, W. E., et al., Nat.New Biol., 236, 176 (1972); Ward, R. A., et al., Proc.Helminthol.Soc.Wash., 39, 525 (1972). The immunizing doses were of the order of a total of 1.times.10.sup.5 sporozoites, a dose smaller than that previously determined necessary for successful immunization in the rodent system. Later results demonstrated that extensive or total protection could be achieved by administration of a total of 4.times.10.sup.7 to 1.7.times.10.sup.8 sporozoites over a period of 9.5 to 13.5 months, Chen, D., Ph.D. thesis, New York University School of Medicine, (1974). Protection against an otherwise lethal sporozoite-induced infection has also been obtained in a certain proportion of Rhesus monkeys immunized with irradiated sporozoites of P. knowlesi (Gwadz at al., Bull. WHO Suppl., 1, 165-173).
In humans, five cases of successful immunization against P. falciparum through the bite of infected irradiated mosquitoes have been reported, Clyde, D. R., et al., Am.J.Med.Sci., 266, 169 (1973); Rieckmann, K., et al., Trans.R.Soc.Trp.Med.Hyg., 68, 258 (1974). The protection was fully effective against a number of geographic isolates of P. falciparum. No cross-reactivity with P. vivax was observed. However, one of the volunteers was subsequently also immunized against sporozoites of P. vivax. Protection in both cases lasted only for a period of a few months.
In addition to the principal facts regarding immunization with sporozoites, there has been developed in the prior art a substantial body of information of a fundamental nature, relating to the nature of the interaction between sporozoites and antibody, and the development of quantitative methods of measurement. Two methods of measurement are significant.
The circumsporozoite (CSP) reaction is observed under phase contrast light microscopy when mature infective sporozoites are incubated with antiserum. Viable sporozoites develop at their posterior end a threadlike precipitate which increases progressively in length, Vanderberg, J., et al., (1969), supra. The CSP reaction can be used to quantitate the antibody response of animals vaccinated with sporozoites, and can also be used to measure the relative infectivity of different sporozoite preparations. The existence of a CSP reaction in a vaccinated animal can in general be correlated with protective immunity; in the primate systems, lack of CSP activity indicates lack of protective immunity but in some instances in rodents, protection has been observed in the absence of CSP reaction.
The sporozoite neutralization reaction (SNA) is carried out by preincubating active sporozites with an antibody preparation, then challenging test animals with the anitbody-treated sporozoites. In primates the SNA reaction correlates well, both positively and negatively, with immunity. However, the tests are time-consuming and expensive to perform since test animals must be challenged, then monitored for the occurrence of patent malarial infection. The criteria for protection may be either complete resistance to an intravenous challenge with viable sporozoites or partial resistance resulting in delayed patency or an altered course of infection.
Standard techniques for measuring antigen antibody reactions are also applicable for the detection and quantitation of anti-sporozoite antibodies, including, for example, radioimmunoassay, immune precipitation, direct and indirect immunofluorescence reactions, and the like.
In addition to the foregoing prior art relating to the establishment of immunity to malarial infections and the nature of the immune reactions, the present invention employs a variety of techniques known in the art, including the preparation of monoclonal antibodies, and recombinant DNA methods. Such methods are described in detail or by reference in the detailed description and examples sections.